Proceedings of the National Academy of

Sciences (PNAS) of the United States of America

Structure of the Decorated Ciliary Doublet Microtubule

Authors:  Meisheng Ma,1 Mihaela Stoyanova,2 Griffin Rademacher,3 Susan K. Dutcher,2 Alan Brown,3 and Rui Zhang,1.

1Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, School of Medicine, St. Louis, MO, USA

2Department of Genetics, Washington University in St. Louis, MO, USA

3Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

IN BRIEF:

Visualizing axonemal microtubules and the proteins that decorate them, on the outside and inside, points to how the underlying periodic architecture supports cilia function.

SUMMARY:

The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells.  In humans, impaired axoneme function causes a range of ciliopathies.  Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components.  Single-particle cryo-electron microscopy is used to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs).  The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility.  This work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.

This study was conducted with IRRF support – Alan Brown, PhD, 2018 and 2019.

Dr. Brown is a member of the faculty of the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School in Boston.  He holds a B.Sc. in Biochemistry from the University of Warwick, Coventry, UK; and a PhD in Biochemistry from the University of Cambridge, Cambridge, UK

Collaborations That Provide Sustained Research Funding

Today’s vision scientists face many funding challenges making it imperative that all support options are available to them.  Similarly, in order to ensure continued funding for young scientists who are developing their independent research projects, the IRRF must maximize every dollar. The formation of partnerships and collaborations with outstanding institutions has made it possible to accomplish this while producing a collective impact.  Since 2013, New York-based Fight For Sight (FFS) and the IRRF have combined resources to provide an annual funding award:  FFS – The International Retinal Research Foundation Grant-in-Aid Award that is offered and administered by FFS.

2018 — Jordan Greco, PhD, University of Connecticut, for his work in the characterization of an ion-mediated protein-based retinal implant.

Dr. Greco obtained his PhD in physical chemistry at the University of Connecticut in 2015.  Under the mentorship of Dr. Robert Birge, Dr. Greco’s graduate thesis work primarily involved the investigation of the structure and function of photoactive proteins, using both spectroscopic and quantum mechanical approaches.  Much of his work has contributed towards the application of the protein bacteriorhodopsin into photonic and biomimetic devices, such as protein-based optical memories and processors, photovoltaic cells, and the retinal implant developed by LambdaVision, Inc.  Concurrent with his work on bacteriorhodopsin, Dr. Greco has contributed to numerous computational analyses for the excited state of behavior of heterocyclic conjugated compounds, (e.g., porphyrin, chlorins, and corroles), carotenoids (e.g., peridinin), and other polyene-based chromophores rooted in biological systems.  Dr. Greco has presented this work to international audiences and he continues to remain active in the field via several multidisciplinary collaborations. (Reprinted from Crunchbase: www.crunchbase.com/person/jordan-greco)

2017 — John T. Pena, MD, PhD, Weill Cornell Medical College for his work in diabetic retinopathy.

Grant Title:  Human ocular fluid contains an intercellular communication system of endogenous exosomes.

Summary:   The vitreous humor of the eye is a clear gel-like structure comprised of collagen and water and fills the back of the eye.

Traditional thinking has been that the vitreous is biologically inactive.  Dr. Pena’s study showed a dense organized network of extracellular vesicles (EVs) in the human vitreous.  However, attempts to image vitreous EVs in whole mount or tissue sections resulted in no evidence of EVs.  Yet, electron microscope (EM) studies and nano-particle tracking analysis proved that millions of EVs exist in the vitreous.  To solve this discrepancy and visualize the native anatomy of vitreous EVs a hypothesis emerged that the nanometer sized EVs were lost during tissue processing secondary to reversible formalin-fixation.  Therefore, this team developed an innovative fixation technique to enable visualization of vitreous EVs in situ.  In addition to identifying the vitreous EVs, it was proven that vitreous EVs are a highly potent vector that can be loaded with synthetic siRNAs or proteins, and subsequently transfects retinal cells in vitro and in vivo.  The team has shown that vitreous EVs can be used as a vector to efficaciously deliver therapeutic recombinant proteins to tissues like the retina and choroid.

Current and Future Academic Plans: Dr. Pena’s academic plans are to continue to grow and become a productive physician-scientist.  He is currently an Assistant Professor of Ophthalmology at Weill Cornell Medicine and Principal Investigator of the laboratory.  Dr. Pena plans to use his training from the clinic and basic sciences to ask pertinent questions that remain a challenge in vision research.  He hopes to provide straightforward solutions that can be translated to benefit his patients and will take the next few years as an opportunity to develop strong academic relationships with his mentors and students.

Related Story:

Since joining the research group of Dr. Robert Birge in 2009, University of Connecticut, Jordan Greco has been actively involved in the research and development that has led to the creation of the protein-based retinal implant and the commercialization of this technology through LambdaVision.  (LambdaVision is led by University of Connecticut alumni and former students in Dr. Birge’s research group, Nicole Wagner, PhD and Jordan Greco, PhD.)

Dr. Greco’s graduate thesis work influenced the design and development of the retinal implant construct and the manufacturing techniques used to produce the prosthetic.  As Chief Scientific Officer, Dr. Greco is responsible for manufacturing the retinal implants and establishing standard operating procedures and quality control measures.  Moreover, his research efforts helped to direct critical proof-of-concept experiments that investigated the efficacy of the retinal implant architecture.

Recently, the company’s robotic system to manufacture films that could cure blindness was brought to the International Space Station U.S. National Laboratory by the SpaceX Dragon spacecraft.

On Earth, it takes LambdaVision approximately five days for each of its three robotic stations to produce an implant.  The process involves a series of alternating dipping steps, which are subject to the effects of gravity.  Once complete, the process results in a membrane approximately 60 um (micrometers) thick.  A micrometer is one-millionth of a meter.

In the weightless conditions of the International Space Station, LambdaVision anticipates producing a more homogeneous and stable film. If successful, Wagner and Greco anticipate they can generate a similar signal with fewer layers of protein.  This would drastically decrease the time for manufacturing, and save on the cost of materials.

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